1. Field of the Invention
This invention relates to methods for accessing microbial diversity through disruption of microbial quorum sensing systems. In particular, this invention enables the isolation of novel microorganisms by dis-enabling quorum sensing systems that are used to maintain microbial cell density at a low level.
2. Description of Related Art
With recent developments in PCR technology and comparative microbial genome sequencing, it has been demonstrated in many environments that the number of microorganisms that have been cultured represents only a percentage of those present in a particular environment. It has been estimated that only approximately 1-5% of existing microorganisms have been cultured in the laboratory.
The organisms which remain “uncultivated” represent a potentially large pool of genes comprising novel microbial diversity. Accessing this diversity would allow the identification for example of enzymes exhibiting novel or enhanced biocatalytic characteristics, novel cofactors, or other novel secondary metabolites such as pharmaceuticals, polymers or other chemicals. Many industrial processes utilize (or could utilize) microbial processes or components thereof and could thus benefit greatly from the isolation of novel microorganisms exhibiting unique characteristics. In addition, novel microorganisms responsible for disease states could be identified. Also, environmental bioremediation could benefit greatly from the identification of microorganisms exhibiting novel biodegradation or bioconversion processes.
Many companies have recently been formed to access microbial diversity by using recombinant techniques to circumvent the inability to culture microorganisms. These techniques are limited in that they can only access single genes or small clusters of genes encoding short metabolic or biosynthetic pathways. The ability to cultivate a microorganism in the laboratory would provide a tremendous advantage.
In nature, bacteria communicate with one another in order to coordinate the expression of specific genes in a cell density-dependent manner. This bacterial communication is called quorum sensing, and it allows bacteria to control gene expression in response to the level of a diffusible signaling molecule called an autoinducer. The signaling molecule binds to a receptor protein, which then activates gene expression. Processes which are controlled by quorum sensing include virulence, bioluminescence, biofilm formation, swarming, sporulation, conjugal transfer of plasmids, and development of competence.
Three main types of quorum sensing systems have been described in bacteria: Type 1, Type 2 and peptide-based. Type 1 quorum sensing has thus far only been demonstrated in Gram negative microorganisms and utilizes acyl homoserine lactones as signaling molecules. Type 2 has been demonstrated in both Gram positive and Gram negative microorganisms and is believed to utilize 4-hydroxy-5-methyl-2H-furan-3-one or 4,5-dihydroxy-2-cyclopenten-1-one as the signaling molecule. Peptide-based quorum sensing systems have been demonstrated only in Gram positive microorganisms and rely on short peptides for gene activation. In addition, other chemical signals have been shown to be used for quorum sensing; these include gamma butyrolactone in Streptomyces sp. and 2-heptyl-3-hydroxy-4-quinolone in Pseudomonas aeruginosa. 
Type 1 quorum sensing utilizes acyl homoserine lactones (AHSL) as signaling molecules. AHSL chemical signals consist of a lactone ring attached to an acyl chain by means of a peptide bond. The acyl chain length is specific for a given microorganism or for an AHSL-mediated process carried out by that microorganism. Some AHSLs contain a carbonyl or hydroxyl group at the 3 position of the acyl chain (e.g., 3-oxo-hexanoyl homoserine lactone, 3-hydroxy-butanoyl homoserine lactone). The paradigm for Type 1 quorum sensing is the Vibrio fischeri luxI/luxR system. The luxI protein catalyzes the synthesis of the autoinducer 3-oxo-hexanoyl homoserine lactone (OHHL). As the cell density increases the autoinducer accumulates and when a threshold level is reached, the OHHL signal interacts with the luxR protein. The luxR/OHHL complex binds to DNA at the lux box resulting in transcription of the bioluminescence genes. Other microorganisms exhibiting Type 1 quorum sensing possess analogs of luxI and luxR.
WO 01/85664 is incorporated in its entirety for its description of Type 2 quorum sensing. Biosynthesis of the Type 2 autoinducer is believed to proceed through progressive steps from methionine through S-adenosyl methionine to S-adenosyl homocysteine to S-ribosyl homocysteine to 4-hydroxy-5-methyl-2H-furan-3-one or 4,5-dihydroxy-2-cyclopenten-1-one. Enzymes involved in the synthesis are believed to include methionine adenosyl transferase, methyl transferase, nucleosidase and the luxS protein or its analogs, which synthesizes 4-hydroxy-5-methyl-2H-furan-3-one or 4,5-dihydroxy-2-cyclopenten-1-one from its precursor. In Vibrio harveyi, the receptors for the Type 2 autoinducer are luxP and luxPQ. When autoinducer concentrations reach a threshold level, the autoinducer interacts with the receptor and luxO is dephosphorylated (and inactivated), thereby preventing activation of a repressor and allowing luxR to activate transcription of the luxCDABE genes.
Many Gram positive bacteria use secreted peptides as autoinducers. Generally, in peptide based quorum sensing systems, the peptide is secreted by an ATP-binding cassette (ABC) transporter. The concentration of the autoinducer increases with cell density, and at a threshold level two component sensor kinases detect the autoinducer. A phoshorylation cascade is initiated which results in phosphorylation of a cognate response regulator protein. The response regulator is thus activated, allowing it to bind DNA and affect transcription of the quorum-sensing regulated genes.
In nature microorganisms regulate microbial processes in response to environmental conditions. In environments, for example, where nutrients are uniformly distributed, it is conceivable that microorganisms regulate cell division such that a high cell density is never achieved and cells remain dispersed; an example of such an environment is the ocean. It is possible that microorganisms utilize quorum sensing to control their cell division, and thus many microorganisms from these environments would thus far have been uncultivable in the laboratory due to quorum sensing. Therefore, if quorum sensing were disrupted or dis-enabled, novel microorganisms from these environments could be cultivated in the laboratory.